C-jun mRNA Research Articles

Abstract 54: MUC1-C regulates PD-L1 expression in acute myeloid leukemia, via downregulation of miRNAs

Abstract Acute myeloid leukemia (AML) is a lethal hematologic cancer in which the tumor microenvironment is characterized by a state of immune dysregulation that fosters disease progression. The PD-L1/PD-1 pathway confers a negative costimulatory signal that induces T-cell exhaustion, supports immune evasion by malignant cells, and is a critical component of the immunosuppressive milieu in AML. While PD-L1 expression in AML is dynamic, little is known about the mechanisms responsible for regulating PD-L1 expression in AML. MUC1 is a heterodimeric oncoprotein aberrantly expressed in solid tumors and hematologic malignancies including AML, which supports critical aspects of the malignant phenotype including cell proliferation, self-renewal, and resistance to apoptosis. Given its critical function is supporting the malignant phenotype of AML cells and its presence on leukemia stem cells, we sought to explore its role in mediating the immunosuppressive milieu of the tumor microenvironment. In the present study, we have demonstrated that suppression of MUC1-C expression via MUC1-specific shRNA or CRISPR/Cas9 gene editing in cell lines, or with a peptide inhibitor of MUC1 signaling (GO-203) in primary patient AML samples, results in the near abrogation of PD-L1 expression as shown by flow cytometry and immuno-blotting. Interestingly, no decrease in PD-L1 mRNA expression was detected by qPCR, suggesting a post-transcriptional regulation of PD-L1 expression. Noncoding RNAs have been shown to regulate critical aspects of oncogenic phenotype through the selective binding to target mRNAs and regulation of protein translation. The microRNAs miR200c and miR34a demonstrate homology with the 3'UTR section of PDL-1 mRNA. Mir200c was recently shown to downregulate the expression of PD-L1 protein in a lung cancer model. Indeed, inhibition of MUC-1 in AML cell lines and patient samples leads to a marked increase in miR200c and miR34a levels. Moreover, ectopic overexpression of these miRNAs in MOLM-14 and THP-1 resulted in near abrogation of PD-L1 expression. To examine if the increase in miR200c and miR34a was representative of a class effect on miRNAs, a miRNA NanoString array was performed in MUC1-silenced MOLM-14 and THP-1 cell lines. MUC1-silenced MOLM-14 cells showed an increase in 786/801 (98.1%) and 698/801 (87.1%) of miRNAs probed in the respective cell lines. miRNAs are processed from precursor (pre-) miRNAs to mature miRNAs by the proteins DICER and Argonaut-2. To elucidate the mechanism of MUC1-C regulation of miRNA expression, MUC1-silenced MOLM-14 and THP-1 cells were evaluated for their expression of pre-miR200c and pre-miR34a. The results demonstrated no change in the miRNA precursor levels, in contrast to the levels of mature miRNAs. This suggested that MUC1-C might regulate the processing of precursor miRNAs to their mature form. MUC1-silenced MOLM-14 and THP-1 demonstrated an increase in the mRNA and protein expression of DICER, but not Argonaut-2. To elucidate the mechanism of MUC1-C regulation of DICER expression, we examined transcription factors known to regulate DICER. cJUN is a key regulator of DICER expression that has been reported to be repressed by MUC1 signaling. MUC1-silenced MOLM-14 and THP-1 cells demonstrated an increase in c-JUN mRNA levels and protein expression. Furthermore, inhibition of cJUN activity in AML cell lines using a peptide inhibitor of c-JUN abrogated DICER expression and restored PD-L1 expression in a dose dependent manner. Moreover, immuno-blotting of MUC1-silenced MOLM-14 and THP-1 cells demonstrated increased expression of total and phosphorylated JNK and ERK, both well known to be mediators of cJUN phosphorylation and transcription, respectively. In conclusion, these findings strongly suggest MUC1-C as a key regulator of microRNA expression and demonstrate a critical mechanism by which MUC1-C signaling exploits noncoding RNAs to promote PD-L1 expression, resulting in an immunosuppressive characteristic of AML cells. Citation Format: Dina Stroopinsky, Athalia Rachel Pyzer, Eleni Anastasiadou, Hasan Rajabi, Abigail Washington, Ashujit Tagde, Maxwell Coll, Leandra Cole, Kristen Palmer, Poorvi Somaiya, Rebecca Karp Leaf, Myrna Nahas, Arie Apel, Salvia Jain, Robin Joyce, Jon Arnason, Frank Slack, Donald Kufe, Jacalyn Rosenblatt, David Avigan. MUC1-C regulates PD-L1 expression in acute myeloid leukemia, via downregulation of miRNAs [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 54.

Differentiation of induced pluripotent stem cells into neural stem cells induced by brain-derived neurotrophic factor via Wnt/β-catenin and extracellular signal-regulated kinase/mitogen-activated protein kinases signal pathway

Objective: To investigate the mechanism of brain-derived neurotrophic factor (BDNF) promoting induced pluripotent stem cells (iPSCs) to differentiate into neural stem cells (NSCs) via Wnt/β-catenin and extracellular signal-regulated kinase/mitogen-activated protein kinases (ERK/MAPK) signal pathways. Methods: iPSCs were cultured and identified. The iPSCs were induced to differentiate into NSCs by BDNF and retinoic acid (RA). Nestin was detected by immunofluorescence and flow cytometry after iPSCs differentiated. The technique of small interfering RNA (siRNA) was used to silence the gene expression of β-catenin and ERK, and iPSCs were divided into control group, BDNF group (adding 10 μg/L BDNF), siRNA-ERK/BDNF group (transfected with siRNA-ERK and adding 10 μg/L BDNF) and siRNA-β-catenin/BDNF group (transfected with siRNA-β-catenin and adding 10 μg/L BDNF). Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of key elements of Wnt/β-catenin and ERK/MAPK signaling pathways, included β-catenin, ERK1/2, c-fos, c-jun, and c-myc. The least significant difference test was used when data were compared between groups. Results: The immunofluorescence showed that iPSCs expressed octamer-binding transcription factor-4 (Oct4), SRY-related HMG box protein-2 (Sox2) and Nanog genes. The flow cytometry showed that Nestin-positive cells were 78.7% for BDNF and 43.5% for RA, and it was only 7.8% for routine medium. Compared with those in the control group, the mRNA expression of β-catenin, ERK1/2, c-fos, c-jun, and c-myc in the BDNF group were upregulated significantly (t=2.80, 2.318, 2.255, 1.799, 1.582, 1.663, all P<0.05), and the same results were acquired with the protein expression (t=2.805, 2.318, 2.255, 1.799, 1.582, 1.663, all P<0.050). Compared with those in BDNF group, the mRNA and protein expression of ERK1/2 in siRNA-ERK/BDNF group down-regulated obviously (t=1.917, 2.042, 1.673, 1.540, all P<0.05), and the mRNA and protein expression of c-fos and c-jun were down-regulated (t=1.022, 0.907, 0.848, 0.801, all P<0.05). However, the mRNA and protein expression of β-catenin and c-myc were not suppressed by siRNA-ERK (t=0.216, 0.185, 0.097, 0.112, all P>0.05). In siRNA-β-catenin/BDNF group, the mRNA and protein expression of β-catenin and c-myc was obviously down-regulated when compared with those in BDNF group (t=3.104, 2.774, 2.235, 1.911, all P<0.05), and expression of ERK1/2, c-fos and c-jun were down-regulated too (t=0.776-1.192, all P<0.05). Conclusion: BDNF promotes the differentiation of iPSCs by activating Wnt/β-catenin and ERK/MAPK signal pathway, there should be cross-talk between the two signal pathways, and c-fos and c-jun may be common nuclear transcription factors.

Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation

Anthrax is a life-threatening disease caused by infection with Bacillus anthracis, which expresses lethal factor and the receptor-binding protective antigen. These two proteins combine to form anthrax lethal toxin (LT), whose proximal targets are mitogen-activated kinase kinases (MKKs). However, the downstream mediators of LT toxicity remain elusive. Here we report that LT exposure rapidly reduces the levels of c-Jun, a key regulator of cell proliferation and survival. Blockade of proteasome-dependent protein degradation with the 26S proteasome inhibitor MG132 largely restored c-Jun protein levels, suggesting that LT promotes degradation of c-Jun protein. Using the MKK1/2 inhibitor U0126, we further show that MKK1/2-Erk1/2 pathway inactivation similarly reduces c-Jun protein, which was also restored by MG132 pre-exposure. Interestingly, c-Jun protein rebounded to normal levels 4 h following U0126 exposure but not after LT exposure. The restoration of c-Jun in U0126-exposed cells was associated with increased c-Jun mRNA levels and was blocked by inactivation of the JNK1/2 signaling pathway. These results indicate that LT reduces c-Jun both by promoting c-Jun protein degradation via inactivation of MKK1/2-Erk1/2 signaling and by blocking c-Jun gene transcription via inactivation of MKK4-JNK1/2 signaling. In line with the known functions of c-Jun, LT also inhibited cell proliferation. Ectopic expression of LT-resistant MKK2 and MKK4 variants partially restored Erk1/2 and JNK1/2 signaling in LT-exposed cells, enabling the cells to maintain relatively normal c-Jun protein levels and cell proliferation. Taken together, these findings indicate that LT reduces c-Jun protein levels via two distinct mechanisms, thereby inhibiting critical cell functions, including cellular proliferation.

Effects of Src on cervical cancer cells proliferation and apoptosis through ERK signal transduction pathway

Objective: To explore the effect of Src on cervical cancer cells through ERK signal transduction pathway. Methods: Experimental study was carried out in vitro. Cervical cancer cell lines Hela (HPV-positive) and C33A (HPV-negative) were treated with Src kinase inhibitor PP2. Then, the cell cycle and apoptosis of each group were evaluated by using flow cytometry (FCM). Western blotting and Real-time PCR were used to detect the levels of the expression of ERK 1/2, c-Fos and c-Jun mRNA and protein respectively. The database was established and analyzed with SPSS statistical software (version 20.0). Results: After down-regulating Src, the cell proliferation was inhibited and cell apoptosis was induced. The proportions of G0/G1 stage of Hela and C33A cell in cell cycle increased while G2/M and S stages decreased. Meanwhile, the mRNA levels of ERK 1, ERK 2, c-Fos and c-Jun increased. And the expression levels of ERK 1/2, phosphorylated ERK 1/2 (p-ERK 1/2) and phosphorylated c-Fos (p-c-Fos) protein decreased, while c-Jun and phosphorylated c-Jun (p-c-Jun) protein expression increased. In addtion, the change level of Hela cell, p-ERK 1/2 and c-Fos protein were lower than that of C33A cell before and after the Src inhibition. Conclusions: Src, involved in regulating the expression of key factors of the ERK signal transduction pathway including p-ERK 1/2 and p-c-Fos, might be capable of promoting the proliferation of cervical cancer cells and inhibiting their apoptosis. The infection with HPV might have adjustable effect on this process.

Elucidation of protective efficacy of Pentahydroxy flavone isolated from Madhuca indica against arsenite-induced cardiomyopathy: Role of Nrf-2, PPAR-γ, c-fos and c-jun

BackgroundMadhuca indica J. F. Gmel. (Sapotaceae) is widely used ethnobotanically as anti-diabetic, antipyretic, hepatoprotective, anti-inflammatory and analgesic. It was shown to possess potent anti-apoptotic property. The aim of the studyTo evaluate the possible mechanism of action of isolated phytoconstituent from Madhuca indica Leaves methanolic extract (MI-ALC) on arsenic-induced cardiotoxicity in rats. Materials and methodsThe 3,5,7,3′,4′-Pentahydroxy flavone (QTN) was isolated and characterized by using HPTLC, 1H NMR, and LC–MS from MI-ALC. QTN (5, 10 and 20mg/kg, p.o.) was administered in arsenic intoxicated rats (5mL/kg, p.o.) for 28days and evaluated for various behavioral, biochemical, molecular and ultra-histological changes. ResultsTreatment with QTN (10 and 20mg/kg, p.o.) significantly inhibited (p<0.05) arsenic-induced electrocardiographic, hemodynamic and left ventricular function alterations. Elevated levels of cardiac markers (LDH, CK-MB, AST, ALT, and ALP), altered lipid metabolism (total cholesterol, triglyceride, LDL, HDL, and VLDL) was significantly restored (p<0.05) by QTN. It also significantly inhibited (p<0.05) altered cardiac oxido-nitrosative stress, Na-K-ATPase level and mitochondrial enzymes (I–IV) activity after arsenite administration. QTN significantly increased (p<0.05) myocardial Nrf-2, PPAR-γ and significantly decreased (p<0.05) myocardial c-fos and c-jun mRNA expressions. Flow cytometric analysis showed that treatment with QTN (10 and 20mg/kg) significantly inhibited (p<0.05) arsenite-induce ROS and apoptosis. It also reduced ultra-histological aberrations induced by sodium arsenite. ConclusionAdministration of 3,5,7,3′,4′- Pentahydroxy flavone (i.e. Quercetin (QTN)) isolated from MI-ALC showed significant protection against arsenic-induced oxido-nitrosative stress and myocardial injury via modulation of Nrf2, PPAR-γ, and apoptosis.

Correlation between the expression of activator protein-1 (c-Fos/c-Jun) mRNA and gingival inflammation

Objective To study the correlation between the expression of activator protein-1 (c-Fos/c-Jun) mRNA and gingival inflammation, so as to discuss the pathogenesis of periodontitis. Methods The gingival tissues were divided into three groups according to the gingival index (GI), including GI=0 group (control group, 14 cases), GI=1 group (15 cases) and GI=2 group (11 cases). The total RNA in each gingival tissue was extracted, and cDNA was synthesized by reverse transcription synthesis. The expressions of c-Fos and c-Jun mRNA in healthy gingival tissue (GI=0 group) were detected by reverse transcription-polymerase chain reaction. The levels of c-Fos and c-Jun mRNA in all the groups were detected by real-time quantitative PCR. Results Both c-Fos and c-Jun mRNA was expressed in healthy gingival tissues. The levels of c-Fos and c-Jun mRNA in GI=1 group was 15.58±9.19 and 3.47±1.77, respectively, which was significantly higher than 1.31±1.03 and 1.32±0.94 in GI=0 group, and the differences were statistically significant (all P 0.05). Conclusions Activator protein-1 (c-Fos/c-Jun) is associated with the degree of gingival inflammation, suggesting that it is involved in the occurrence and development of gingival inflammation. Key words: Activator protein-1; c-Fos; c-Jun; Gingival inflammation; Gingival index

Effect of ulinastatin pretreatment on endoplasmic reticulum stress during myocardial injury in patients undergoing mitral valve replacement with cardiopulmonary bypass

Objective To evaluate the effect of ulinastatin pretreatment on endoplasmic reticulum stress during myocardial injury in the patients undergoing mitral valve replacement (MVR) with cardiopulmonary bypass (CPB). Methods One hundred patients of both sexes, aged 35-64 yr, weighing 40-80 kg, of American Society of Anesthesiologists physical status Ⅱ or Ⅲ (New York Heart Association Ⅱ or Ⅲ), scheduled for elective MVR with CPB, were divided into ulinastatin pretreatment group (UP group, n=50) and normal saline control group (NS group, n=50) using a random number table.Ulinastatin 0.5×104 U/kg was intravenously infused over 1 h before skin incision, and administration was repeated every 4 h until the end of operation in group UP, while the equal volume of normal saline was given instead in group NS.Immediately after opening the right atrium (T0), at 30 min after aortic clamping (T1) and while suturing the right atrium (T2), blood samples were collected from the radial artery for measurement of the concentrations of plasma creatine kinase-MB and cardiac troponin T by enzyme-linked immunosorbent assay.Right auricle specimens were obtained after blood sampling at each time point for determination of the expression of glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein and c-Jun N-terminal kinase protein and mRNA (by real-time polymerase chain reaction and Western blot, respectively) and apoptosis in cardiomyocytes (by TUNEL). The apoptosis rate was calculated. Results Compared with group NS, the plasma concentrations of creatine kinase-MB and cardiac troponin T at T1 and T2 and apoptosis rate at T2 were significantly decreased, and the expression of glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein and c-Jun N-terminal kinase protein and mRNA was down-regulated at T1 and T2 in group UP (P<0.05). Conclusion The mechanism by which ulinastatin pretreatment inhibits apoptosis in cardiomyocytes and attenuates myocardial injury is related to decrease in endoplasmic reticulum stress in the patients undergoing MVR with CPB. Key words: Trypsin inhibitors; Apoptosis; Cardiopulmonary bypass; Heart value prosthesis implantation; Myocardium; Endoplasmic reticulum; Stress

Pentosan polysulfate inhibits IL-1β-induced iNOS, c-Jun and HIF-1α upregulation in canine articular chondrocytes.

Osteoarthritic (OA) chondrocytes are shown to express inducible nitric oxide synthase (iNOS) which produces high concentrations of nitric oxide (NO), particularly when stimulated with proinflammatory cytokines. NO is involved in OA cartilage degradation. On the other hand, c-Jun N-terminal Kinase (JNK) pathway mediates the activation and transcription of c-Jun, which is required for interleukin-1 (IL-1)-induction of matrix metalloproteinases-13 (MMP-13) in OA pathogenesis. Therefore, the selective inhibition of iNOS and c-Jun is a promising target for treatment and prevention of OA. The purpose of the study was to investigate the inhibitory effects of pentosan polysulfate (PPS) on IL-1β-induced iNOS, c-Jun and HIF-α isoforms upregulation in canine articular chondrocytes (CACs). Primary (P0) chondrocytes were isolated and cultured from femoral head cartilages of three (3) dogs. First passage (P1) chondrocytes were preincubated with 0, 1, 5, 15 and 40 μg/mL of PPS for 4 hr before treatment with 10 ng/mL rhIL-1β for a further 8 hr. In addition, we evaluated the effects of single and multiple cytokine with or without LPS on iNOS protein induction. PPS significantly inhibited (P < 0.05) IL-1β-induced iNOS, c-Jun and HIF-1α mRNA upregulation in a dose-dependent pattern. iNOS mRNA was significantly inhibited at 15 and 40 μg/mL whereas c-Jun and HIF-1α were significantly downregulated at 5, 15 and 40 μg/mL of PPS compared to chondrocytes treated with only rhIL-1β. Intriguingly, CACs were recalcitrant to single IL-1β, TNF-α or LPS-induction of iNOS protein including to a combination of IL-1β+TNF-α, IL-1β+LPS except to TNF-α+LPS and IL-1β+TNF-α+LPS suggestive of a protective mechanism from iNOS detrimental effects on perpetuating OA. IL-1β+TNF-α+LPS-induced iNOS protein expression was significantly abrogated by PPS. We demonstrate for the first time that PPS is a novel inhibitor of IL-1β-induced iNOS, c-Jun, and HIF-1α mRNA upregulation and iNOS protein induction which may be beneficial for prevention and treatment OA.

The AP-1 Transcription Factor c-Jun Promotes Arthritis by Regulating Cyclooxygenase-2 and Arginase-1 Expression in Macrophages.

Activation of proinflammatory macrophages is associated with the inflammatory state of rheumatoid arthritis. Their polarization and activation are controlled by transcription factors such as NF-κB and the AP-1 transcription factor member c-Fos. Surprisingly, little is known about the role of the AP-1 transcription factor c-Jun in macrophage activation. In this study, we show that mRNA and protein levels of c-Jun are increased in macrophages following pro- or anti-inflammatory stimulations. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment cluster analyses of microarray data using wild-type and c-Jun-deleted macrophages highlight the central function of c-Jun in macrophages, in particular for immune responses, IL production, and hypoxia pathways. Mice deficient for c-Jun in macrophages show an amelioration of inflammation and bone destruction in the serum-induced arthritis model. In vivo and in vitro gene profiling, together with chromatin immunoprecipitation analysis of macrophages, revealed direct activation of the proinflammatory factor cyclooxygenase-2 and indirect inhibition of the anti-inflammatory factor arginase-1 by c-Jun. Thus, c-Jun regulates the activation state of macrophages and promotes arthritis via differentially regulating cyclooxygenase-2 and arginase-1 levels.

Correlation between Loss of E-cadherin, Matrix-metalloproteinases and c-Jun expression in Oral Carcinogenesis

Background: Earlier invitro studies have observed that loss of E-cadherin is responsible for progression and metastasis of cancer by upregulation of c-Jun protein. There being lack of simultaneous evaluation of matrix metalloproteinase (MMP) -2 and -9, E-cadherin and c-Jun mRNA and protein.&#x0D; Objective: This study aimed to correlate the above-mentioned parameters to evaluate the pathway of oral carcinogenesis.&#x0D; Methods: The study included 100 controls, 50 patients with oral precancerous conditions (OPC) and 100 oral cancer patients. MMPs were evaluated by gelatin zymography, ECAD and CJUN mRNA and protein expression by semi quantitative RT-PCR and western blot respectively.&#x0D; Results: The levels of active and total MMP-2 and MMP-9 were significantly higher in patients with OPC and oral cancer patients as compared to controls. A significant increase in truncated E-cadherin and c-Jun protein was observed in malignant tissues as compared to adjacent normal tissues while CJUN mRNA levels were comparable. Higher values of c-Jun protein and MMPs, and lower values of ECAD mRNA were associated with reduced overall survival. A positive correlation was observed between truncated E-cadherin, MMPs and c-Jun protein.&#x0D; Conclusions: MMPs modulate cell-cell adhesion by increasing truncation of E-cadherin resulting in loss of E-cadherin which is responsible for upregulation of c-Jun protein in oral cancer.

C-jun is differentially expressed in embryonic and adult neural precursor cells.

c-jun, a major component of AP-1 transcription factor, has a wide variety of functions. In the embryonic brain, c-jun mRNA is abundantly expressed in germinal layers around the ventricles. Although the subventricular zone (SVZ) of the adult brain is a derivative of embryonic germinal layers and contains neural precursor cells (NPCs), the c-jun expression pattern is not clear. To study the function of c-jun in adult neurogenesis, we analyzed c-jun expression in the adult SVZ by immunohistochemistry and compared it with that of the embryonic brain. We found that almost all proliferating embryonic NPCs expressed c-jun, but the number of c-jun immunopositive cells among proliferating adult NPCs was about half. In addition, c-jun was hardly expressed in post-mitotic migrating neurons in the embryonic brain, but the majority of c-jun immunopositive cells were tangentially migrating neuroblasts heading toward the olfactory bulb in the adult brain. In addition, status epilepticus is known to enhance the transient proliferation of adult NPCs, but the c-jun expression pattern was not significantly affected. These expression patterns suggest that c-jun has a pivotal role in the proliferation of embryonic NPCs, but it has also other roles in adult neurogenesis.

HOXC10 up-regulation promotes gastric cancer cell proliferation and metastasis through MAPK pathway.

As an important regulator of embryonic morphogenesis, homeodomain-containing gene 10 (HOXC10) has been found to promote progression of human cancers and its expression indicates poor survival outcome. However, very few studies are available on the role of HOXC10 in gastric carcinoma. Therefore, the aim of this study was to determine the role of HOXC10 in gastric cancer and the potential mechanism underlying its function for cancer biology. A primary gastric cancer mouse model was obtained via intra-gastric wall injection of gastric cancer cells and was used to evaluate the function of HOXC10 during gastric cancer progression in vivo. Immunohistochemistry was performed to visualize and measure HOXC10 protein expression in gastric cancer tissue. Cells were transfected with plasmids to increase the expression of HOXC10, and siRNA transfection was performed to suppress HOXC10 expression. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting were utilized to measure mRNA and protein expression, respectively. Proliferation, migration, and invasion were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound healing assay, and matrigel invasion assay in vitro, respectively. HOXC10 expression was significantly increased in gastric cancer tissues compared to matched normal tissues. HOXC10 up-regulation significantly increased tumor volumes in nude mice. Plasmid transfection significantly increased HOXC10 protein and mRNA expressions and effectively promoted cell proliferation. Moreover, HOXC10 up-regulation significantly promoted migration and invasion of gastric cancer cells. Mechanistic investigation showed that HOXC10 up-regulation significantly increased mRNA and protein expression of mitogen-activated protein kinase (MAPK) signaling related genes, including c-myc, c-jun and p53, while also modulating the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and P38 but not their total protein levels. This study demonstrated the tight link between HOXC10 and gastric cancer cell proliferation and metastasis via involvement of the MAPK pathway.

C-Jun N-terminal kinase promotes stem cell phenotype in triple-negative breast cancer through upregulation of Notch1 via activation of c-Jun.

c-Jun N-terminal kinase (JNK) plays a vital role in malignant transformation of different cancers, and JNK is highly activated in basal-like triple-negative breast cancer (TNBC). However, the roles of JNK in regulating cancer stem-like cell (CSC) phenotype and tumorigenesis in TNBC are not well defined. JNK is known to mediate many cellular events via activating c-Jun. Here, we found that JNK regulated c-Jun activation in TNBC cells and that JNK activation correlated with c-Jun activation in TNBC tumors. Furthermore, the expression level of c-Jun was significantly higher in TNBC tumors than in non-TNBC tumors, and high c-Jun mRNA level was associated with shorter disease-free survival of patients with TNBC. Thus, we hypothesized that the JNK/c-Jun signaling pathway contributes to TNBC tumorigenesis. We found that knockdown of JNK1 or JNK2 or treatment with JNK-IN-8, an adenosine triphosphate-competitive irreversible pan-JNK inhibitor, significantly reduced cell proliferation, the ALDH1+ and CD44+/CD24- CSC subpopulations, and mammosphere formation, indicating that JNK promotes CSC self-renewal and maintenance in TNBC. We further demonstrated that both JNK1 and JNK2 regulated Notch1 transcription via activation of c-Jun and that the JNK/c-Jun signaling pathway promoted CSC phenotype through Notch1 signaling in TNBC. In a TNBC xenograft mouse model, JNK-IN-8 significantly suppressed tumor growth in a dose-dependent manner by inhibiting acquisition of the CSC phenotype. Taken together, our data demonstrate that JNK regulates TNBC tumorigenesis by promoting CSC phenotype through Notch1 signaling via activation of c-Jun and indicate that JNK/c-Jun/Notch1 signaling is a potential therapeutic target for TNBC.

Three Arachidonoylamide Derivatives Inhibit Pro-Inflammatory Genes Expression by Modulating NF-kB and AP1 Activities.

Since the anti-inflammatory activity of arachidonic acid derivatives was previously reported, we synthesized three new amide derivatives of arachidonic acid (AA-Ds) and tested their anti-inflammatory effects on an in vitro skin inflammation model. Aim of our study was to find derivatives of natural compounds able to down regulate inflammatory signal transduction pathway. Human keratinocytes cell line (HaCaT) was cultured and induced by cytokines in the presence of AA-Ds. Cytokines administration elicited an inflammatory response mediated by NF-kB and STAT-1 activation that induced proinflammatory genes expression. By real time PCR we found that 24 hours after induction all AA-Ds significantly inhibit inducible Nitric Oxide Synthase (iNOS), TNFα, Inhibitor α of NF-kB, chemokine (C-X-C motif) ligand 9 and 10 genes expression. We analyzed their molecular effects in particular on the iNOS gene expression. Since iNOS transcript half-life did not change with AA-Ds treatment, we excluded a prominent role of post-transcriptional regulation for this gene and focused our attention on its transcriptional regulation. Starting three-five hours after cytokines induction, HaCaT cells, pretreated with each compound, showed inhibition of both NF-kB DNA-binding and NF-kB p65-Ser536 phosphorylation. STAT1 activation was inhibited only by AA-D4 derivative. To explain why the inhibition of iNOS expression began late after induction we analyzed activities of others key transcription factors. AA-Ds treatment elicited early increases of AP1 DNA binding as well as c-Jun, c-Fos and Fra-1 mRNA levels. Our data agree with the repressing effects of AP1 on human iNOS promoter previously described in others cell systems (Kleinert et al.). AA-Ds shown to be good candidates as inhibitors of several pro-inflammatory genes induction and our study provides indications for their possible use as new antiinflammatory drugs.

MicroRNA 10b promotes abnormal expression of the proto-oncogene c-Jun in metastatic breast cancer cells.

MicroRNAs have been shown to act as oncogenes or tumor suppressers via various cellular pathways. Specifically, in breast cancer, upregulation of miR-10b is positively associated with aggressiveness of tumors. However, the mechanism by which miR-10b contributes to cell malignancy is largely unknown. Here we show that at the receiving end of the miR-10b pathway is the proto-oncogene c-Jun, a transcription factor that plays a critical role in stimulation of cell proliferation and tumor progression. c-Jun is known to be translationally activated by loss of cell contacts or restructuring of the cytoskeleton. A comprehensive analysis of miRNA expression exhibited a significant increase in miR-10b expression. This was supported by analysis of breast cancer cells, which showed that loss of E-cadherin in metastatic cells is accompanied by elevation of miR-10b and interestingly, by a marked increase in accumulation of c-Jun. Silencing miR-10b in metastatic breast cancer cells leads to a decline in c-Jun expression, whereas overexpression of miR-10b in HaCaT cells is sufficient to elevate the accumulation of c-Jun. The increase in c-Jun protein accumulation in metastatic cells is not accompanied by an increase in c-Jun mRNA and is not dependent on MAPK activity. Knockdown and overexpression experiments revealed that the increase is mediated by NF1 and RhoC, downstream targets of miR-10b that affect cytoskeletal dynamics through the ROCK pathway. Overall, we show the ability of miR-10b to activate the expression of c-Jun through RhoC and NF1, which represents a novel pathway for promoting migration and invasion of human cancer cells.

RNA-binding protein HuR and the members of the miR-200 family play an unconventional role in the regulation of c-Jun mRNA.

Post-transcriptional gene regulation is a fundamental step for coordinating cellular response in a variety of processes. RNA-binding proteins (RBPs) and microRNAs (miRNAs) are the most important factors responsible for this regulation. Here we report that different components of the miR-200 family are involved in c-Jun mRNA regulation with the opposite effect. While miR-200b inhibits c-Jun protein production, miR-200a tends to increase the JUN amount through a stabilization of its mRNA. This action is dependent on the presence of the RBP HuR that binds the 3′UTR of c-Jun mRNA in a region including the mir-200a binding site. The position of the binding site is fundamental; by mutating this site, we demonstrate that the effect is not micro-RNA specific. These results indicate that miR-200a triggers a microRNA-mediated stabilization of c-Jun mRNA, promoting the binding of HuR with c-Jun mRNA. This is the first example of a positive regulation exerted by a microRNA on an important oncogene in proliferating cells.

EIF3d is an mRNA cap-binding protein that is required for specialized translation initiation.

Eukaryotic mRNAs contain a 5' cap structure critical for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression1 despite eIF4E inactivation. Here we describe a new cellular cap-dependent pathway of translation initiation that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eukaryotic initiation factor 3 (eIF3) complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modeling of cap recognition by eIF3d. eIF3d makes specific contacts to the cap, as exemplified by cap analog competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs2 such as the cell proliferation regulator c-Jun. The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a new mechanism of cap-dependent translation independent of eIF4E, and illustrate how modular RNA elements work in concert to direct specialized forms of translation initiation.

SPANXA suppresses EMT by inhibiting c-JUN/SNAI2 signaling in lung adenocarcinoma.

SPANXA (Sperm Protein Associated with the Nucleus on the X-chromosome, family members A1/A2) acts as a cancer-testis antigen expressed in normal testes, but dysregulated in various tumors. We found that SPANXA is highly expressed in low-invasive CL1-0 cells compared with isogenous high-invasive CL1-5 cells. SPANXA was preferably expressed in tumor tissues and associated with the prolonged survival of lung adenocarcinomas. SPANXA suppressed the invasion and metastasis of lung cancer cells in vitro and in vivo. By the expression microarray and pathway analysis, we found that the SPANXA-altered genes were enriched in the epithelial–mesenchymal transition (EMT) pathway. SPANXA reduced SNAI2 expression resulted in up-regulating E-cadherin. c-JUN acts as the positive-regulator of EMT. Silencing SPANXA increased c-JUN mRNA expression and blockage of c-JUN led to SNAI2 down-regulation. Our results clearly characterized SPANXA as an EMT inhibitor by suppressing c-JUN-SNAI2 axis in lung adenocarcinoma.

Association of long noncoding RNA and c-JUN expression in hepatocellular carcinoma

ABSTRACTBackground: Long noncoding RNAs(lncRNAs) have emerged as key elements in modulating gene expression in different biological contexts.Patients and methods: We used quantitative real-time PCR (Qpcr) to evaluate the expression of lncRNA-UCA1 and C-JUN in serum of 70 patients with hepatocellular carcinoma (HCC), 32 patients chronic hepatitis C (CHC) and 38 healthy subjects and their correlation with different clinicopathological factors.Results: The expression of lncRNA-UCA1 and C-JUN was positive in 91.4%HCC patients with strong discriminating power between HCC and healthy subjects and CHC patients as well. The median follow up period was 29 months. The survival analysis showed that both lncRNA-UCA1 and C-JUN were independent prognostic factors. Of note, we identified C-JUN expression changes consistent with the lncRNA-UCA1 target regulation.Conclusion: This information sheds light on the possible role of lncRNA-UCA1 and C-JUN mRNA as promising diagnostic and prognostic markers as well as potential therapeutic targets in HCC.

Protective Effects of Minor Components of Curcuminoids on Hydrogen Peroxide-Treated Human HaCaT Keratinocytes.

Hydrogen peroxide, one of the reactive oxygen species (ROS), can cause intracellular oxidative stress associated with skin aging and/or photoaging. Curcumin, a polyphenol in turmeric, has been reported to exhibit biological activity. In this study, five naturally occurring curcuminoids [curcumin, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), monohydroxy-DMC, and monohydroxy-BDMC] were used to investigate their protective roles against hydrogen peroxide-induced oxidative stress in the immortalized human keratinocyte cell lines (HaCaT cells). These five curcuminoids at 10 μM, but not at 5 μM, were shown to exhibit cytotoxicities toward HaCaT keratinocytes. Therefore, a 5 μM concentration of the five curcuminoids was selected for further investigations. Cells were pretreated with or without curcuminoids for 2.5 h before 24-h hydrogen peroxide (150 μM) treatments. Pretreatments with the minor components monohydroxy-DMC or monohydroxy-BDMC, but not curcumin, DMC, and BDMC, showed protective activity, elevating cell viability compared to cells with direct hydrogen peroxide treatments. Pretreatments with monohydroxy-DMC and monohydroxy-BDMC showed the best protective effects, reducing apoptotic cell populations and intracellular ROS, as demonstrated by flow cytometry, as well as reducing the changes of the mitochondrial membrane potential compared to cells with direct hydrogen peroxide treatments. The pretreatments with monohydroxy-DMC and monohydroxy-BDMC reduced c-jun and c-fos mRNA expression and p53 tumor suppressor protein expression and increased HO-1 protein expression and glutathione peroxidase (GPx) activity, respectively, compared to cells with direct hydrogen peroxide treatments. The five curcuminoids exhibited similar hydrogen peroxide-scavenging activity in vitro. It was proposed that monohydroxy-DMC and monohydroxy-BDMC could induce antioxidant defense systems better than curcumin, DMC, or BDMC could against hydrogen peroxide-induced oxidative stress and apoptosis of HaCaT keratinocytes and that they may have potential as ingredients in antiaging cosmetics for skin care.